Intron-containing algal transgenes mediate efficient recombinant gene expression in the green microalga Chlamydomonas reinhardtii

Baier, Thomas; Wichmann, Julian; Kruse, Olaf; Lauersen, Kyle J.

doi:10.1093/nar/gky532

Cited by 145 publications

(190 citation statements)

References 56 publications

Supporting

Mentioning

182

Contrasting

Unclassified

Order By: Relevance

“…Compared to BKT from other organisms, the CrBKT contains a long peptide extension on its C-terminus (Figure 1c), which appears to be an unfolded peptide region. Here, we took the CrBKT sequence and used our recently developed transgene optimization strategy (Baier et al, 2018b) to enable its re-integration into the algal nuclear genome and overexpression. When CrBKT was expressed, it caused the green algal cells to noticeably change colour from green to red, indicating the conversion of carotenoids to ketocarotenoids, with astaxanthin representing a major component of these.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, we demonstrated that transgenes could be optimized for expression from the algal nuclear genome by codon optimization and systematic incorporation of the first intron of the C. reinhardtii RuBisCO small subunit II gene to mimic host regulatory structures (Baier et al, 2018b). We have recently used this strategy for overexpression of numerous foreign genes (Lauersen et al, 2016;Lauersen et al, 2018;Wichmann et al, 2018) , as well as re-coding and overexpression of the endogenous photodecarboxylase (CrFAP) (Yunus et al, 2018) for various metabolic engineering activities in this host.…”

Section: Intragenic Expression Of β-Ketolase In C Reinhardtiimentioning

confidence: 99%

“…The BKT (AY860820.1) found within the genome of C. reinhardtii was synthetically redesigned with codon optimization and intron spreading as recently described (Baier et al, 2018b). The synthetic optimized CrBKT gene coding sequence (CDS) was deprived of the last 345 bp and a small region was added to make a Cterminal GSG linker prior to optimization and the synthetic gene produced by GeneArt (Germany).…”

Section: Design and Cloning Of Expression Cassettesmentioning

confidence: 99%

“…Pigments were extracted with acetone from the digested fraction and spectra recorded. 100 HS UVM4 0,95 ± 0,09 a 2,70 ± 0,21 a ----0,14 ± 0,01 a 3,8 ± 0,5 a 7,5 ± 1,3 a 21,4 ± 3,9 a -4,3 ± 0,2 a -bkt 0,19 ± 0,04 b 1,81 ± 0,14 b,c 0,38 ± 0,01 a 0,75 ± 0,02 a 27,2 ± 2,6 a 51,7 ± 5,7 a 0,19 ± 0,02 b 1,5 ± 0,2 b 1,2 ± 0,2 b 8,9 ± 1,5 b -2,1 ± 0,1 b 21,3 ± 2,3 a 41,6 ± 4,1 a npq2 0,67 ± 0,20 c 2,49 ± 0,67 b ----0,15 ± 0,02 a --18,6 ± 4,5 a 16,0 ± 4,3 a 5,6 ± 0,5 c --n2bkt 0,25 ± 0,07 b 1,42 ± 0,38 c 0,41 ± 0,06 a 0,76 ± 0,09 a 48,3 ± 8,6 b 87,2 ± 11,4 b 0,14 ± 0,03 a --11,3 ± 2,5 b 3,5 ± 0,9 b 1,8 ± 0,2 d 30,7 ± 10,5 a 56,8 ± 18,3 a 100 TAP UVM4 1,41 ± 0,34 a 2,81 ± 0,11 a ----0,56 ± 0,05 a 4,9 ± 2,1 a 8,7 ± 2,9 a 16,8 ± 1,1 a -5,3 ± 2,1 a -bkt 0,38 ± 0,08 b 2,39 ± 0,09 b 0,42 ± 0,06 a 0,69 ± 0,03 a 45,2 ± 6,9 a 73,2 ± 3,7 a 0,65 ± 0,01 b 1,5 ± 0,6 b 2,1 ± 0,6 b 8,6 ± 0,5 b -1,0 ± 0,3 b 17,4 ± 2,4 a 28,8 ± 1,3 a npq2 1,26 ± 0,49 a 2,80 ± 0,03 a ----0,69 ± 0,04 b --12,3 ± 1,3 c 19,3 ± 2,0 a 4,0 ± 1,2 a --n2bkt 0,72 ± 0,09 c 2,45 ± 0,03 b 0,3 ± 0,01 b 0,62 ± 0,02 b 62,1 ± 1,1 b 123,7 ± 0,8 b 0,65 ± 0,01 b --13,1 ± 1,2 c 1,6 ± 0,2 b 0,6 ± 0,2 b 12,5 ± 0,4 b 25,5 ± 0,8 b 500 HS UVM4 0,87 ± 0,21 a 2,54 ± 0,24 a ---0,14 ± 0,01 a 3,7 ± 0,9 a 7,5 ± 1,8 a 21,2 ± 8,3 a 2,8 ± 0,4 a 4,2 ± 1,1 a -bkt 0,09 ± 0,02 b 0,93 ± 0,09 b 0,44 ± 0,02 a 0,71 ± 0,08 a 27,6 ± 2,8 a 44,9 ± 10,7 a 0,13 ± 0,06 a 2,5 ± 0,5 a 2,0 ± 0,5 b 16,0 ± 5,7 a 3,9 ± 0,7 a,b 2,3 ± 1,0 b 49,1 ± 4,5 a 81,1 ± 12,9 a npq2 0,60 ± 0,24 a 2,12 ± 0,73 a ----0,18 ± 0,04 a --20,2 ± 8,0 a 22,0 ± 4,2 c 4,9 ± 1,6 a,c --n2bkt 0,08 ± 0,03 b 0,66 ± 0,22 b 0,40 ± 0,07 a 0,77 ± 0,04 a 56,9 ± 9,4 b 106,0 ± 8,4 b 0,14 ± 0,03 a --17,2 ± 7,4 a 4,7 ± 2,1 b 2,8 ± 0,4 b,c 44,2 ± 10,1 a 84,2 ± 16,5 a 500 TAP UVM4 1,04 ± 0,17 a 2,51 ± 0,34 a ----0,71 ± 0,05 a 4,8 ± 1,1 a 10,4 ± 2,3 a 17,7 ± 3,2 a 1,6 ± 0,6 a,b 5,4 ± 1,6 a -bkt 0,21 ± 0,03 b 1,80 ± 0,24 b 0,5 ± 0,04 a 0,72 ± 0,02 a 39,5 ± 3,3 a 56,3 ± 6,9 a 0,68 ± 0,03 a,b 1,7 ± 0,4 a 1,8 ± 0,3 b 9,6 ± 1,5 b 1,3 ± 0,4 a 1,2 ± 0,3 b 28,3 ± 5,5 a 40,5 ± 5,5 a npq2 0,98 ± 0,16 a 2,39 ± 0,29 a ----0,64 ± 0,07 b,c --14,7 ± 2,4 c 22,2 ± 5,4 c 5,0 ± 0,2 a --n2bkt 0,47 ± 0,07 c 1,67 ± 0,20 b 0,41 ± 0,04 b 0,7 ± 0,07 a 78,7 ± 9,9 b 131,7 ± 15,6 b 0,62 ± 0,02 c --14,7 ± 2,2 c 2,2 ± 0,5 b 0,9 ± 0,1 b 24,9 ± 5,6 a 42,5 ± 8,9 a car/100 chl CrBKT sequence was built by gene synthesis after in silico design using codon optimization and systematic spreading of the rbcs2 intron 1 sequence to minimize exon lengths as previously described to enable robust transgene expression from the nuclear genome of this alga (Baier et al, 2018b). (c) Schematic overview of all expression vectors used in this work and their respective ketocarotenoid accumulation efficiencies.…”

Section: Extractability and Simulated Digestion Of Ketocarotenoids Inunclassified

See 3 more Smart Citations

Turning a green alga red: engineering astaxanthin biosynthesis by intragenic pseudogene revival in Chlamydomonas reinhardtii

Perozeni

Cazzaniga

Baier

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

The green alga Chlamydomonas reinhardtii does not synthesize high-value ketocarotenoids like canthaxanthin and astaxanthin, however, a β-carotene ketolase (CrBKT) can be found in its genome.CrBKT is poorly expressed, contains a long C-terminal extension not found in homologues and likely represents a pseudogene in this alga. Here, we used synthetic re-design of this gene to enable its constitutive overexpression from the nuclear genome of C. reinhardtii. Overexpression of the optimized CrBKT extended native carotenoid biosynthesis to generate ketocarotenoids in the algal host causing noticeable changes the green algal colour to a reddish-brown. We found that up to 50% of native carotenoids could be converted into astaxanthin and more than 70% into other ketocarotenoids by robust CrBKT overexpression. Modification of the carotenoid metabolism did not impair growth or biomass productivity of C. reinhardtii, even at high light intensities. Under different growth conditions, the best performing CrBKT overexpression strain was found to reach ketocarotenoid productivities up to 4.5 mg L -1 day -1 . Astaxanthin productivity in engineered C. reinhardtii shown here is competitive with that reported for Haematococcus lacustris (formerly pluvialis) which is currently the main organism cultivated for industrial astaxanthin production. In addition, the extractability and bio-accessibility of these pigments was much higher in cell wall deficient C. reinhardtii than the resting cysts of H. lacustris. Engineered C. reinhardtii strains could thus be a promising alternative to natural astaxanthin producing algal strains and may open the possibility of other tailor-made pigments from this host.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Intragenic Expression Of β-Ketolase In C Reinhardtiimentioning

confidence: 99%

Section: Design and Cloning Of Expression Cassettesmentioning

confidence: 99%

Section: Extractability and Simulated Digestion Of Ketocarotenoids Inunclassified

See 2 more Smart Citations

Turning a green alga red: engineering astaxanthin biosynthesis by intragenic pseudogene revival in Chlamydomonas reinhardtii

Perozeni

Cazzaniga

Baier

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…The nucleotide sequence of C. reinhardtii Hy5 (Uniprot: A0A2K3DRN7) was optimized as previously described (Baier et al, 2018) using the Intronserter tool (Jaeger et al, 2019) (https://bibiserv.cebitec.uni-bielefeld.de/intronserter). The codon optimized, intron containing gene was chemically synthesized (GenScript, Piscataway Township, New Jersey) and cloned between Bam HI- Bg lII or Eco 32I- Eco RI in the pOpt2_mVenus_Paro vector (Wichmann et al, 2018).…”

Section: Methodsmentioning

confidence: 99%

Ubiquitin ligase component LRS1 and transcription factor CrHy5 act as a light switch for photoprotection inChlamydomonas

Lämmermann

Wulf

Chang

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

25Survival under excess light conditions requires the light-induced accumulation of protein 26 LHCSR3 and other photoprotection factors, to enable efficient energy-dependent 27 quenching in the green microalga Chlamydomonas reinhardtii. Here, we demonstrate 28 that the high light-tolerant phenotype of mutant hit1 is caused by a de-repression of 29 promoters belonging to photoprotection genes, which in turn results from an inactivation 30 of the E3 ubiquitin ligase substrate adaptor LRS1. Transcriptome analyses of hit1 31 revealed massive alterations of gene expression modulation as a consequence of 32 perturbed LRS1 function, indicating its role as a crown regulator. In conjunction with 33 random forest-based network modeling, these transcriptome analyses predicted that 34 LRS1 controls photoprotection gene expression via an algal HY5 homolog as its prime 35 transcription factor target. CrHY5 binds to T-box elements present in the promoters of 36 these genes and its inactivation in the hit1 mutant via CRISPR-Cas9 genome editing, 37 confirmed the regulatory connection between LRS1 and CrHY5, predicted by the 38 network analysis. 39 40 41 42 43 44 45 46 47 48 54 Light provides the energy driving photosynthesis and it is an important cue controlling 55 many physiological processes in phototrophic organisms. In the seed plant Arabidopsis 56 thaliana the E3 ubiquitin ligase substrate adaptor COP1/SPA is a master switch of light 57 signaling, by repressing the accumulation of various transcription factors required for the 58 induction of light-dependent processes like photomorphogenesis or flowering in 59 darkness. Repression is achieved by ubiquitination of target transcription factors and 60

show abstract

Successful reversal of transgene silencing in Chlamydomonas reinhardtii

Beauchemin,

Merindol,

Fantino

et al. 2023

Biotechnology Journal

View full text Add to dashboard Cite

Chlamydomonas reinhardtii has been successfully engineered to produce compounds of interest following transgene integration and heterologous protein expression. The advantages of this model include the availability of validated tools for bioengineering, its photosynthetic ability, and its potential use as biofuel. Despite this, breakthroughs have been hindered by its ability to silence transgene expression through epigenetic changes. Histone deacetylases (HDAC) are main players in gene expression. We hypothesized that transgene silencing can be reverted with chemical treatments using HDAC inhibitors. To analyze this, we transformed C. reinhardtii, integrating into its genome the mVenus reporter gene under the HSP70‐rbcs2 promoter. From 384 transformed clones, 88 (22.9%) displayed mVenus positive (mVenus+) cells upon flow‐cytometry analysis. Five clones with different fluorescence intensities were selected. The number of integrated copies was measured by qPCR. Transgene expression levels were followed over the growth cycle and upon SAHA treatment, using a microplate reader, flow cytometry, RT‐qPCR, and western blot analysis. First, we observed that expression varies with the cell cycle, reaching a maximum level just before the stationary phase in all clones. Second, we uncovered that supplementation with HDAC inhibitors of the hydroxamate family, such as vorinostat (suberoylanilide‐hydroxamic‐acid, SAHA) at the initiation of culture increases the frequency (% of mVenus+ cells) and the level of transgene expression per cell over the whole growth cycle, through histone deacetylase inhibition. Thus, we propose a new tool to successfully trigger the expression of heterologous proteins in the green algae C. reinhardtii, overcoming its main obstacle as an expression platform.This article is protected by copyright. All rights reserved

show abstract

Intron-containing algal transgenes mediate efficient recombinant gene expression in the green microalga Chlamydomonas reinhardtii

Cited by 145 publications

References 56 publications

Turning a green alga red: engineering astaxanthin biosynthesis by intragenic pseudogene revival in Chlamydomonas reinhardtii

Turning a green alga red: engineering astaxanthin biosynthesis by intragenic pseudogene revival in Chlamydomonas reinhardtii

Ubiquitin ligase component LRS1 and transcription factor CrHy5 act as a light switch for photoprotection inChlamydomonas

Successful reversal of transgene silencing in Chlamydomonas reinhardtii

Contact Info

Product

Resources

About